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Jason Sheltzer @jsheltzer.bsky.social · Sep 7

Very cool!

1) did you make any attempt to eliminate guides likely to cause false-positives due to chromosome truncations? (PMID: 38811841)

2) are you including guides targeting new genes discovered from T2T sequencing, particularly on the sex chromosomes? (PMID: 37612512)

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Next up - we want to improve the tumor-specific accumulation of CDK11 to bypass this toxicity, and we’re looking for other emerging drug targets to create mouse models for. If you’re interested in collaborating, feel free to reach out!

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Along the way, we also learned a ton about the biology of CDK11, the 1p36 locus (one of the most frequently-deleted regions across cancer genomes!), and the CDK-dependent control of gene expression.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

If you have a mutation that blocks the interaction between your drug and its target, and so long as that mutation is tolerated in mice, then you can do the same thing that we did - make a mouse with the resistance mutation and see what happens after drug treatment.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

I think that this approach can substantially improve the drug development process. Nearly all cancer drugs fail during clinical testing, and toxicity is one of the most common reasons why. We urgently need better approaches to predict and study toxicity in a preclinical setting.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

We injected the G568S mice with a mouse cancer cell line and then treated them with a high dose of MEL-495R (which was tolerable to the G568S mice but toxic to WT mice). This resulted in a significant anti-cancer effect, verifying that on-target toxicity limits effective dosing.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

This suggested that toxicity (which we believed to be CDK11-dependent) was limiting our ability to effectively dose these mice. To verify this, we returned to our CDK11-G568S mouse strain to tease apart the cause.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

This gave us confidence to move forward with the drug. We identified a non-toxic dose of MEL-495R and tested it in several xenografts. However, it showed very little anti-cancer activity. A splicing qPCR indicated that this non-toxic dose wasn’t appreciably inhibiting CDK11.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

We bred a large cohort of CDK11-mutant (G568S) and CDK11-WT mice, treated them with an ultra-high dose of MEL-495R, and it worked beautifully. The wild-type mice became very sick while the CDK11-G568S mice were totally fine. Our drug is specific for CDK11 - in living mice!

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

We thought - if the mice expressing this mutation are still affected by our CDK11 inhibitor, then that tells us that it’s causing CDK11-independent toxicity. In contrast, if these mice are resistant to the drug, then any side effects of the drug in WT mice are due to CDK11.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

We came up with a way to answer this question. We had discovered a mutation in CDK11 that blocks drug binding to it. We thought - what if we put that mutation into a mouse? So, we found the mouse ortholog of the human mutation, CRISPR’d it into some zygotes, and did exactly that.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

You can throw every biochemical assay in existence against a drug, but that won’t do it - we can’t test all ~20,000 human proteins at once, it’s really hard to determine drug concentrations in each tissue, and in vivo drug metabolism can generate dozens of derivative compounds.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

This brought us to an issue that is absolutely crucial for cancer drug development. All cancer drugs have at least some toxicity. If you have a drug against a new target (like CDK11), how do you know if that toxicity is due to CDK11 inhibition or due to something else?

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Now, we wanted to take the drug in vivo. Unfortunately, it had a terrible ADME profile. We worked with the talented chemists at Meliora to develop an improved CDK11 inhibitor, and we created MEL-495R, which exhibits potent CDK11 inhibition and superior PK properties.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

This makes CDK11 a new “CYCLOPS” gene, as described by @RameenBeroukhim and Bill Hahn - a vulnerability established when a gene is deleted so that only one copy of that gene remains: www.cell.com/fulltext/S00...

Cancer Vulnerabilities Unveiled by Genomic Loss

Reducing the mRNA levels of genes that have suffered copy number loss due to genomic instability in cancer cells leads to tumor-cell-specific growth inhibition. Thus, probing cancer genomes for genes ...

www.cell.com

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Next, we figured out why - 1p36 is where CDK11 and its activating cyclin (cyclin L) are encoded. Having a lower dosage of these genes enhances the dependency on the remaining enzyme, creating a synthetic-lethal relationship.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

For any cancer therapy, finding a biomarker to predict sensitivity is key. We analyzed screening data with CDK11-targeting CRISPR, CDK11-targeting RNAi, and OTS964 treatment, and they all pointed to the same biomarker: Chr1p36 deletions enhance sensitivity to CDK11 ablation.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Next, we uncovered the cell biology of CDK11 inhibition. We found that CDK11 controls gene expression through two distinct mechanisms: ensuring accurate splicing via SF3B1, and, separately, controlling the transcription of certain genes by promoting the activation of RNAPII.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

First, we did the fundamental molecular genetics: targeting CDK11 with OTS964 or CRISPR killed cancer cells, and this could be rescued with a mutation in CDK11 that blocked drug binding or with gRNA-resistant cDNA (but not if the cDNA contained a kinase-inactivating substitution).

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

CDK11 is a member of the cyclin-dependent kinase family. Other CDK inhibitors, like the CDK4/6 inhibitor palbociclib, have revolutionized the treatment of certain cancer subtypes. So, we set out to characterize CDK11 as a possible therapeutic vulnerability in cancer.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

One of these mischaracterized drugs is called OTS964. While it was initially developed as a PBK inhibitor, we showed that it actually functions by inhibiting CDK11 - making it the first-ever inhibitor of this poorly-characterized kinase.

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

To back up, my lab is deeply interested in trying to improve the cancer drug development process. We’ve shown that many “targeted” cancer therapies function via off-target mechanisms and kill cells independently of their putative targets -

x.com/JSheltzer/st...

Jason Sheltzer on X: "Our new paper is out today. We used CRISPR to uncover some really striking findings with several drugs and drug targets in clinical trials. Also, we accidentally found the first-ever inhibitor of the cyclin-dependent kinase CDK11. https://t.co/Rthvr67cyk" / X

Our new paper is out today. We used CRISPR to uncover some really striking findings with several drugs and drug targets in clinical trials. Also, we accidentally found the first-ever inhibitor of the cyclin-dependent kinase CDK11. https://t.co/Rthvr67cyk

x.com

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Link here: www.biorxiv.org/content/10.1...

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches t...

www.biorxiv.org

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Jason Sheltzer @jsheltzer.bsky.social · Aug 4

Thrilled to share our new paper describing the development and characterization of CDK11 inhibitors for cancer therapy. We also establish a new system that I think represents a huge leap forward in our ability to understand drug toxicity in a living organism.

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Jason Sheltzer @jsheltzer.bsky.social · Jun 4

At Stanford, I’ll be affiliated with the Stanford Cancer Institute, Radiation Oncology, and Pathology. My lab will be in the Research Park complex on Page Mill Road. I’m looking forward to getting to know my new community! Feel free to reach out and say hi.

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